Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchial smooth muscle. The major naturally occurring substances that induce bronchial smooth muscle contraction are ligands of G-protein-coupled receptors (GPCRs), such as allergen proteases, thrombin, and those contained in allergen-IgE activated mast cell granules (e.g. histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin). In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions and muscle fiber shortening. In contrast, ligands acting on G-alpha-s-coupled receptors, such as albuterol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyper-responsiveness (AHR), suggesting that underlying abnormalities in structural cells including airway smooth muscle (ASM) cells contribute to the asthmatic diathesis. Dysregulation of procontractile, GPCR signaling in ASM could mediate enhanced contractility. 10-15% of people with asthma experience severe, life threatening attacks and even death despite aggressive treatment with bronchodilators and corticosteroids. Nearly half of these (10-20 million) are sensitized (i.e. have IgE-mediated allergy) to filamentous fungi (e.g. Aspergillus fumigatus, Af), which has been designated severe asthma with fungal sensitization (SAFS). Current therapies for SAFS including antifungals or omalizumab monoclonal antibody (mAb) targeting IgE have not achieved uniform success. Utilizing a model of allergic airway inflammation in mice induced by respiratory Af exposure, we have 2 overarching aims for this project: 1) identify derangements in ASM contraction signaling downstream of inflammatory mediators; 2) examine the functions of allergen protease activity in AHR, particularly in relation to allergen-ASM interactions. Protease activity is a common and important feature of allergens capable of inducing asthma, most notably from ubiquitous fungi such as Af. Whether any allergens affect ASM contraction directly has never been explored. Previously we found a causal link between fungi and asthma occurring independently of allergenicity; in other words, host inflammatory response to the allergen. The secreted Af protease Alp1 was detected in the airways of asthmatic subjects but not controls. In mouse and in vitro studies, we found that Alp1 directly promoted AHR by degrading ECM components, leading to dysregulation of ASM contraction. In fiscal year 19, we demonstrated that Alp1 from Aspergillus fumigatus can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrated that Alp1 directly increased contractile force. The second area of emphasis for this project is the study of Regulators of G protein signaling (RGS) proteins in the lung. RGSs bind to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by catalyzing their intrinsic GTPase activity and by blocking downstream effector interactions. Although they are generally considered to act as negative regulators of GPCR signaling pathways, the physiological function of RGS proteins in the lung is mostly unknown. We identified expression of several RGS proteins (RGS4, RGS5) in bronchial smooth muscle of humans and mice. In fiscal year 19, we examined the phenotype of mice with global and smooth muscle-specific Rgs4 gene deletion. Nearly 15% of patients with severe asthma develop bronchospasm following ingestion of aspirin and other non-steroidal anti-inflammatory drugs, a condition known as aspirin-exacerbated respiratory disease (AERD), which may result in part from disordered metabolism of arachidonic acid metabolites such as prostaglandin E2 (PGE2). We also examined the role of RGS4 in AERD by challenging AERD-like (ptges1-/-) mice with aspirin. We found that PGE2 secretion is a negative homeostatic signal within the lung epithelium to allergen-induced injury, and we identify RGS4 as a critical NSAID-inducible factor in respiratory epithelial cells that impairs this host response and increases susceptibility to bronchospasm. RGS4 expression in lung epithelium is increased in subjects with severe asthma, with expression that correlates strongly with the degree of functional impairment. Allergen-induced AHR was significantly diminished in Rgs4-/- mice, a finding that was dependent on increased airway PGE2 levels. RGS4 inhibited allergen-induced PGE2 secretion in human bronchial epithelial cells, and treatment with the RGS4 inhibitor CCG203769 in WT or (ptges1-/-) mice alleviated AHR induced by allergen and/or aspirin challenge, respectively, in association with increased PGE2 production. We conclude that RGS4 is a potential biomarker and interventional target in severe and aspirin-associated asthma. In collaboration with Dr. Krishnan and colleagues, we propose high throughput screening to identify inhibitors of Alp1 protease. Such compounds will be used to study the functions of protease allergens in AHR and elucidate ASM contraction mechanisms in fungal-associated asthma. Eventually, we hope to identify small molecules targeting Alp1 protease for the treatment of patients with SAFS. Toward this end, we have generated recombinant Alp1 protease in mammalian cells to use for compound screening.